Human prolactin (hPRL) is a member of a single-domain 4-helix bundle cytokine family. hPRL activates the prolactin receptor (hPRLr) by sequentially binding the extracellular portions of two hPRLrs. hPRLr is a complex protein containing multiple domains that include two domains in the extracellular portion of the molecule. Binding of hPRL creates a dimeric hPRLr complex involving up to seven structurally-distinct binding interfaces. The extracellular domains of hPRLr independently articulate and both bind hPRL, increasing the reaction's complexity. An increasing body of evidence suggests that hPRL is a viability factor necessary for the growth and survival of cells of the breast, prostate, and immune system. This has increased interest in rationally engineered agonists and antagonists. Such protein engineering requires maps of the functional epitopes within the binding sites and a detailed understanding of the mechanics of binding. Neither the kinetics nor energetics of the binding reactions been determined, and the roles played by specific residues of either hPRL or hPRLr in the functional chemical topologies of these interfaces are not understood. This gap in our knowledge is the primary barrier to our ability to rationally engineer these proteins. Therefore, we propose to use several newly-developed approaches to identify the contributions of individual residues to hPRL/hPRLr binding and biological activity, as well as determine the mechanistic details of binding. To this end, we propose the following Specific Objectives: 1) Identify the functional epitopes and map the energetic contributions in sites 1 and 2 of hPRL when binding to the extracellular domain of the hPRLr; 2) Identify the functional epitopes and map the energetic contributions of the extracellular domains of hPRLr when binding to either site 1 or site 2 of hPRL, or binding at the receptor/receptor interface; 3) Determine the binding kinetics and energetics of the isolated extracellular domains of the hPRLr and hPRL; and 4) Correlate structurally-based kinetic and energetic changes in ligand/receptor binding with in vivo changes in receptor activation and biological activity. These studies will be the first to define the functional chemical topology of each binding interface of hPRL/hPRLr and to identify which binding interfaces provide the interactions necessary to position the intercellular domains in spatial positions that allow their activation by the Janus kinase.